Cytogenetic Examination of Leukemia.pptx

leukimia

1. Cytogenetic Examination of Leukemia

2. Cytogenetics  study of genetic material at cellular level mainly concerned with studies of chromosomes Non-random chromosomal abnormalities are a common feature of many hematolymphoid disorders and are a key component of their pathogenesis. Since the discovery of Philadelphia chromosome in 1960, chromosomal abnormalities had been studied more extensively in cancer patients, including leukemia.

3. chromosome analysis is critical in the laboratory workup of most known or suspected myelodysplastic / myeloproliferative disorders, leukemias can provide diagnostic confirmation; information useful for classification, staging, and prognostication; information to guide appropriate choice of therapy; evidence of remission or relapse

4. WHO Classification of Tumors of Hematopoietic and Lymphoid Tissues ( 2008) categorizes hematolymphoid neoplasms based on: clinical and biological features morphology immunophenotype cytogenetic abnormalities and molecular genetic mutations.  some myeloid and many lymphoid disorders demonstrate chromosomal abnormalities

5. CHROMOSOMES and the CELL CYCLE (a) G1-S-G2 : interphase (b) interphase Mitosis : (c) early prophase (d) early metaphase (e) late metaphase (f) Anaphase (g) telophase DNA replication :S-phase (S = synthesis) Cells in G1 : Go cycle

6. Chromosome abnormality Numerical : Euploidy : triploidy , tetraploidy Aneuploidy Structural : balanced and unbalanced translocation deletion inversion insertion

7. TERMINOLOGY Karyotype Nomenclature Translocation (t) Insertion (ins)

8. TERMINOLOGY Karyotype Nomenclature Inversion (inv) [180 o of chromosome segment] Deletion (del) [terminal or inter- stitial ]

9. Clonal abnormality consists of two or more cells with the same chromosome gain or structural rearrangement, or three cells with the same chromosome loss. If a single-cell abnormality is identified, the process cannot be defined as clonal ; however, if it is a characteristic abnormality associated with a specific hematolymphoid disorder or is observed in a patient that demonstrated it as part of an abnormal clone in a previous study, an extended workup is indicated. Sometimes, an apparently balanced rearrangement not known to be associated with any hematolymphoid disorder will be observed. This observation necessitates the examination of a PHA-stimulated peripheral blood culture to determine if the abnormality is constitutional in nature.

10. Chromosome rearrangements in leukemia - AML Disorder Chromosome rearrangement Genes involved FAB classification AML with t(8;21) t(8;21)(q22;q22) AML1-ETO M2 AML with inv(16) or t(16;16) Inv(16)(p12;q22), t(16;16)(p13;q22) CBF β ; MYH11 M4Eo APL t(15;17)(q22;q12) PML-RAR α M3 AML with 11q23 (MLL) abnormalities t(9;11)(p21;q23), t(11;19)(q23;p13.1), t(11;19)(q23;p13.3) AF9;-MLL MLL-ENL M5 AML with multilineage dysplasia -5, del(5q), -7, del(7q), +8, del(11q), del(20q), +21, translocation involving 3q21 and 3q26, t(9;22)(q34;q11.2) M1 M6

11. Chromosome rearrangements in leukemia - CMPD Disorder Chromosome rearrangement Genes involved CML (chronic phase) t(9;22)(q34;q11.2) BCR-ABL CML (accelerated or blast phase) +8, I(17q), +19, +Ph’ Polycythemia vera +8,+9, del920q), del(13q) Chronic idiopathic nyelofibrosis +8, -7, del(7q), del(11q), del(13q), del(20q) Essential thrombocythemia +8, del(13q)

12. Chromosome rearrangements in leukemia – ALL & CLL Disorder Chromosome rearrangement Genes involved FAB classification Precurspr B-lymphoblastic leukemia or lymphoma t(1;19)(q23;p13.3) t(9;22)(q34; q11.2) 11q23 rearrangements PBX-E2A BCR-ABL MLL ALL-L1/L2 Precurspr T-lymphoblastic leukemia or lymphoma Rearrangements at 14q11.2, 7q35 and 7p14-15 del)9p) Translocations at 1p32 T-cell receptor loci CDKN2A TAL1 ALL-L1/L2 CLL +12, del13q, del11q23-24, 14q+, del17p13

13. Prognostic factors in AML Cytogenetics Favourable : t(15;17), t(8;21), inv(16), NPM mutation, CEBPA mutation Intermediate : normal karyotype , other non-complex changes Infavourable : del(5), del(7), abnormal (3q), t(6;11), t(10;11), t(9;22), complex rearrangements (>3 unrelated abnormalities), FLT3 internal tandem duplications (FLT3-ITD). Bone marrow response to remission induction : favourable if <5% blast after 1 st course Age: >60 yrs unfavourable

14. Prognostic significance of cytogenetic in ALL Poor prognosis t(9;22)(q34;q11.2)/ BCR-ABL t(11q23;V)/ MLL gene arrangement Hypodiploid (<45 chromosome) t(1;19)(q23;p13.3)/ PBX1-E2A Good prognosis t(12;21)(p13;q22)/ TEL-AML1 Hyperdiploid

15. Specimen Anticoagulant : preservative-free heparin Bone marrow : tissue of choice for leukemia Peripheral blood : chronic granulocytic leukemia & chronic lymphocytic leukemia Volume: at least 1-2 ml, depending on cell count

16. Reagents Culture media FdU (0.5 x 10 -5 M) Uridine (4 x 10 -4 M) BrdU (2 x 10 -3 M) KCl 0.075 M PBS pH 7.3 Giemsa 10% or Leishman stain Trypsin

17. Culture Culture can be synchronized or unsynchronized. Unsynchronized : Direct, short term (usually 24 h), or overnight Synchronized: cell division blocked at various points in the cell cycle and subsequently released  Synchronization of cell division. Blocking agents: MTX, FdU and FdU+BrdU

18. Advantage of direct culture: provide a result within 24 h Advantage of Short-term (24 h) cultures vs direct: Better metaphase quality and in some cases, clonal rearrangements are detectable only in cultured preparations, such as the diagnostic t(15;17) in acute promyelocytic leukemia. If the specimen is extremely limited in quantity  one culture : short-term unstimulated culture

19. Procedure Medium preparation Culture : set up & synchronize Harvesting Dropping Banding Analyze

20. Culture Culture media RPMI 1640 + L-glutamine + FCS + Pen-Strep Marrow max Cell count/ culture (10 ml) : 10-20 x 10 6 Incubate for 24 hours Synchronize with FdU /Ur and BrdU

21. Culture failure May be due to: Inappropriate specimen : peripheral blood with no circulating blasts wrong anticoagulant Specimen with clots Contaminated specimen Insufficient specimen Dead cells : may be caused by delay in sample transport or specimen was not kept at 4 C and transported in ice if delay is unavoidable. Reagent failure : contamination Human error (usually unlikely).

22. Harvest involves incubating the culture in a mitotic spindle inhibitor, such as Colcemid (0.05 μg /ml) to collect metaphases then adding a hypotonic solution (0.075 M KCl ), and incubating at 37◦C for 10–15 min. Followed by several fixation steps using chilled 3:1 methanol:glacial acetic acid. With each successive fixation, the cell pellet is being “cleaned up,” ensuring an optimal cell suspension with little or no background when slides are prepared. After 4–5 fixation steps, the cells are resuspended in fixative and can be stored at –20◦C until dropping

23. Dropping Goal : prepare slides with wellspread chromosomes that can be recognized as individual “metaphase spreads.” Precleaning microscope slides with 95% ethanol can facilitate uniformity of chromosome spreading and enhance the quality of metaphase preparations. Drop fixed cells on wet microscope slide. Age slide in optimal humidity (45–55% ) and ambient temperature (70–75◦F) Dry in oven (60 o C) overnight.

24. Banding Chromosome morphology is characterized by size, centromere position, and banding pattern. The bands observed in metaphase chromosomes are prepared by processing slides using various different methodologies and staining solutions, including: quinacrine mustard and fluorescence microscopy (Q banding) Giemsa or an equivalent stain (G banding) hot alkali followed by staining with Giemsa or acridine orange (R banding) chromosome denaturation prior to Giemsa staining (C-banding) to visualize heterochromatic DNA ( centromere )

25. Analysis Cytogenetic analysis requirements for neoplastic studies Karyotyping is done according to the ISCN (An international system for human cytogenetic nomenclature). Full karyotyping of 20 cells when possible. If more than one cell line is present, at least one karyotype must be prepared from each cell. The resolution should be at least 300-400 bands (for cancer). If < 20 analysable cells and an abnormality has been detected, the number of abnormal and normal cells (if any) is reported. If < 20 cells can be examined and an abnormality is not detected, the number of cells studied is reported and additional procedures (FISH, molecular studies) may be recommended if clinically appropriate.

26. CML & ALL: t(9:22)(q34;q11.2) From : Micale , MA. Classical and molecular cytogenetic analysis of hematolymphoid disorder. In : Crisan D. Hematopathology . Genomic mechanism of Neoplastic disease, Springer, 2011.

27. AML From : Micale , MA. Classical and molecular cytogenetic analysis of hematolymphoid disorder. In : Crisan D. Hematopathology . Genomic mechanism of Neoplastic disease, Springer, 2011.

28. ALL From : Micale , MA. Classical and molecular cytogenetic analysis of hematolymphoid disorder. In : Crisan D. Hematopathology . Genomic mechanism of Neoplastic disease, Springer, 2011.

29. Result of Karyotyping in Indonesian patients with AML No metaphase: 15/48 Normal karyotype : 18/48 Complex karyotype : 11/48 Clonal abnormalities: 45,XY,-7 46, XX, del (5q) 45, XY, -21 44, XX, -1, -17 Data from study of Syampurnawati M & Akbar DF (ongoing study)

30. Acknowledgments Shelley Tahija , MD, ClinPath Meilani Syampurnawati , MD, PhD Delta Fermikuri Akbar, BSc Theresia Kushandini , BSc

31. Thank you